Arenaviruses are responsible for hemorrhagic fevers with high mortality. Effective therapies against Lassa fever virus (LASV) and Argentine hemorrhagic fever virus (Junin, JUNV) infections are urgently needed to address public health and national security concerns. Intervention strategies directed at the arenavirus envelope glycoprotein (GPC) provide a rational basis for the development of novel antiviral agents. We have recently demonstrated that an interaction between the ectodomains of the GPC fusion subunit (G2) and the unusual stable signal peptide (SSP) is essential for pH-dependent activation of GPCmediated membrane fusion. Interestingly, our preliminary results strongly suggest this interaction is targeted by small-molecule compounds (SIGA Technologies) that act to stabilize the pre-fusion GPC complex against low pH activation, thereby preventing virus entry. Importantly, a prototype inhibitor ST-193 has been shown to protect against lethal LASV infection in guinea pigs. We have established a collaboration with SIGA to capitalize on our knowledge of GPC and these promising lead compounds, in order to identify a broadspectrum arenavirus therapeutic for clinical development. The specific aims of this proposal are: 1. To identify and characterize molecular determinants of the SSP-G2 interface that are responsible for the antiviral activity of SIGA fusion inhibitors. We will examine the contributions of individual sidechains to membrane-fusion activity and its inhibition. We will explore the role of sequence variation in JUNV and LASV in imparting species specificity to these compounds. Drug-resistant isolates derived in cell culture will be used to identify sidechains important for antiviral activity. 2. To characterize structural features of SIGA inhibitors that contribute to their antiviral activity. We will investigate chemical derivatives of lead compounds to define structure-activity relationships that determine potency and breadth of inhibition. We will also capitalize on the structural diversity of four distinct chemical classes of SIGA inhibitors to model a common pharmacophore using computational methods. 3. To evaluate the therapeutic efficacy of selected inhibitors. Optimized, drug-like compounds will be used in guinea pig models of pre-symptomatic JUNV and LASV infection to determine therapeutic efficacy. These studies will be important in the selection of a compound for non-human primate studies in accordance with the FDA Two-Animal Rule for IND filing and clinical development. This work fits within the RMRCE IRF-Viral Therapeutics, and interacts with RP 3.4.